Paroxysmal nocturnal hemoglobinuria (PNH) and idiopathic aplastic anemia (IAA) are closely related disorders: several lines of evidences indicate that both have a T-cell-mediated autoimmune pathogenesis.

We have previously reported that in almost all PNH patients, compared to controls, there is a significant increase of CD8 T cells (CDd1-restricted, GPI-specific T cells), that through their TCR recognize a CD1d dimer loaded with glycosylphosphatidylinositol (GPI); as well as of CD8 T cells producing interferon gamma (IFNγ) in response to GPI presented by CD1d (CDd1-restricted, GPI-reactive T cells). Recently, we have found that GPI specific T cells were also present in about 70% of IAA patients and that only in this subset of AA patients there was also an increased frequency of INFγ producing T cells upon GPI presentation. We have postulated that, through the toxic effect exerted by IFNγ upon the TCR recognition of GPI within the CD1d groove, these cells target selectively normal (GPI-positive) hematopoietic stem cells (HSC), whereas the PNH (GPI-negative) HSC are spared.

Here we first confirm in a new series of 14 PNH patients that the frequency of GPI-specific and GPI reactive T cells increase when peripheral blood mononuclear cells (PBMNC) from PNH patients are grown on antigen presenting cells (APC) expressing CD1d, with the condition that either endogenous or exogenous GPI Is present We have also confirmed that this increase is restricted to CD8 T cells. Next, we have characterized these cells after 7 days of co-culture: we find that GPI specific T cells (CD8+CD1d/GPI dimer+ T cells) and INFγ producing T cells (CD8+INFγ+ T cells) did not express any of the following surface molecules: CD25, IL17, CD56 and CD16.

In all our experiments, INFγ producing T cells outnumber GPI-specific T cells: suggesting that INFγ response is not restricted to GPI-specific T cells (those that are able to recognize the GPI presented by CD1d APCs). In order to dissect the relationship between the two cell types thus defined, after 7 days of co-culture with CD1d APCs competent for GPI synthesis, we have performed simultaneous staining with both GPI-loaded CD1d-dimer and anti-IFNγ. Surprisingly, we found that in none of 20 patients (14 PNH and 6 IAA) were present CD8 T cells stained with both GPI-loaded CD1d-dimer and intracellular anti-IFNγ. CD1d/GPI dimer+ T cells were 0.8±0.32%; INFγ+ T cells were 3.22±0.56%; CD1d/GPI dimer+/INFγ+ T cells were 0.04±0.01% (Fig. 1A). Thus, the T cells that increase specifically after the CD1d mediated GPI presentation consist of two distinct populations. Since their frequency increases in response to GPI stimulus, we surmised that the GPI-specific T cells upon TCR engagement with the GPI loaded CD1d activate in a specific manner a population of CD8 T cell that produce IFNγ. We have tested this hypothesis by comparing in the same setting (co-culture on CD1d APCs either unable or able to synthesize GPI) the PBMNC of 8 PNH patients with and without depletion by immunomagnetic sorting of T cells able to bind the CD1d/GPI dimer: without depletion we observed, as expected, an increased frequency of both GPI-specific and of IFNγ producing T cells; in contrast, with CD1d/dimer+ depleted PBMNC, we observed not only the absence of GPI-specific T cells (confirming that the depletion procedure had been effective), but also a marked reduction of INFγ producing (GPI reactive) T cells (Fig. 1B).

This study indicates that both GPI specific (CD8+ CD1d/GPI dimer+) T cells and INFγ producing (CD8+INFγ+) T cells increase specifically after CD1d-mediated GPI presentation. At the moment we do not yet know their respective significance, but we can consider at least two possibilities: (i) The GPI specific T cells are able by themselves to damage or kill normal (GPI-positive) HSC, whereas the increased number of INFγ producing T cells is essentially an epiphenomenon; (ii) GPI specific T cells, after recognizing through their TCR the GPI presented by CD1d on normal (GPI-positive) HSC, are able to amplify their own cytotoxic action by recruiting a larger population of CD8+ T cells that is thus stimulated to produce INFγ. In conclusion, we have found that at least two distinct but functionally related auto-reactive populations of T cells are involved in the complex pathogenesis of PNH and IAA.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.